Anopheles mosquitoes are the primary vectors of malaria, one of the most deadly and costly diseases in human history. There is no effective vaccine against malaria, although promising candidates are being tested. Current control strategies rely on prompt diagnosis and drug treatment and the reduction of human exposure to infectious mosquito bites. These measures are becoming less effective as insecticide- and drug-resistance increases. Novel approaches to malaria control are urgently needed. Recent application of next-generation sequencing technologies to mosquito genomics offers exciting opportunities to enhance our understanding of mosquito genetics, physiology, and behavior, in ways that were previously unimaginable. This information will enable the development of new strategies to combat malaria and other mosquito-borne diseases. Only female mosquitoes feed on blood and transmit disease pathogens. Anopheles mosquitoes use the XX/XY sex-determination system. There is evidence that a dominant male-determining factor(s) on the Y chromosome initiates sexual differentiation in an Anopheles species. Genetic studies show that insect Y chromosomes are involved in the regulation of male fertility, mating behavior, and global gene expression. However, molecular characterizations of Y genes are rare outside of a few model species, mostly due to the repetitive nature of the Y chromosome and the difficulty of cloning repeat-rich heterochromatic sequences. We have recently discovered a few Y genes including a novel gene we call Guy1 (Gene Unique to Y). We choose to investigate the function of Guy1 in this R21 project because Guy1 is transcribed at the very onset of zygotic transcription in the early embryo and it is a single copy gene with no autosomal or X paralog. We will test the hypothesis that Guy1 is involved in sexual differentiation in the early embryo. We will investigate the effect of ectopic Guy1 expression (Aim 1) and Guy1 knockdown (Aim 2) on sex-related phenotypes as well as general changes in development and embryonic gene expression. This research will contribute to our long-term objective, which is to illuminate the genetic basis of sexual dimorphism in anopheline mosquitoes and develop relevant and new control strategies. The approach developed in this research will inform future work in other Anopheles species. We anticipate extending beyond this R21 to investigate the mechanism of Y gene function, develop mosquito control applications through the manipulation of Y genes, and establish Y genes as novel markers for phylogenetic, population and mating behavior studies.